Templating of solid particles by polymer multilayers

ABSTRACT

Process for encapsulation of an uncharged crystalline solid particle include treating the crystalline solid particle material with amphiphilic substances and subsequently coating the material with a layer of charged polyelectrolyte or coating the material with a multilayer comprising alternating layers of oppositely charged polyelectrolytes.

The invention is directed to (i) the encapsulation of uncharged organicsubstances in polymeric capsules by using a multi-step strategy thatinvolves the introduction of charge to the surface of the microcrystalswith an amphiphilic substance, followed by consecutively depositingpolyelectrolytes of opposite charge to assemble a multilayered shell ofpolymeric material around the microcrystal template, and (ii) theformation of polymer multilayer cages derived from the coated crystalsby facile removal of the crystalline template.

In recent years, microcapsules have received considerable attentionbecause of their technological importance in the fields of medicine,pharmaceutics, agriculture and cosmetics.¹⁻⁷ The vast majority ofapplications are associated with the controlled release of encapsulatedactive ingredients (e.g. drugs, vaccines, antibodies, hormones,pesticides and fragrances) under well-defined conditions. Despite thearray of encapsulation technologies available, including those based onliposomes, microparticles and microemulsions, there has been an intenseinterest in strategies to encapsulate and deliver water-insolublepharmaceutical drugs in stable and aqueous forms.^(8,9) Methods toachieve this have commonly included the incorporation of such drugs intomicelles and microspheres, emulsification of the drug with oils, the useof concentrated solutions of water-soluble polymers, as well assolubilization or suspending the drug with non-ionic detergents. Analternative and recent approach has been to coat water-insolublecrystalline drugs with a membrane lipid, thus allowing dispersion of thecrystal in an aqueous medium.⁹ This represents an elegant method toprepare injectable forms of water-insoluble substances. The advantagesof this process are the significantly higher concentrations (up to 40%w/v) of the injectable drug afforded (compared with other methods), andthe stability of the coated dispersion.

The coupling of self-assembly and colloidal templating provides anelegant and versatile means to encapsulate a variety of functionalmaterials including biological macromolecules and to create core-shellstructures for potential use in the fields of medicine, pharmaceutics,catalysis and separations.¹⁰⁻¹⁸ The method entails coating colloidalparticles dispersed in aqueous media by the nanoscale electrostaticself-assembly of charged polymeric materials. This strategy exploits thefact that the colloidal entities, which serve as the templates, have aninherent surface charge, thus rendering them water dispersible andproviding the necessary charge essential for adsorption of subsequentlayers and polyelectrolyte multilayer encapsulation. Recently, thisapproach has been employed to entrap proteins¹⁸ and construct newclasses of composite colloids.¹¹⁻¹⁷ The colloids employed have rangedfrom charged polymer latices¹¹⁻¹⁷ to biological templates, e.g. cells¹⁰and protein crystals¹⁸.

Solid crystalline organic compounds are an important class of materialsthat are widely employed in pharmaceutics as drugs. The controlledcoating of such compounds is of widespread interest.¹⁹ However, manycrystalline materials that are of significance in medicine, for examplecrystals composed of low molecular weight drugs, are uncharged and havea low solubility in water. For such drugs, their encapsulation andapplication in an aqueous medium often represents a substantial problem.

An object of the invention was therefore to provide a method for theencapsulation of uncharged materials.

The problem underlying the invention is solved by a process for theencapsulation of an uncharged solid particle material comprising

-   (a) treating the solid particle material with an amphiphilic    substance and-   (b) subsequently coating the solid material with a layer of charged    polyelectrolyte or with a multilayer comprising alternating layers    of oppositely charged polyelectrolytes.

The encapsulation of materials by using colloidal templating wasextended to uncharged solid templates, thereby presenting an alternativeand advantageous strategy to other encapsulation methods.

With the process according to the invention it is surprisingly possibleto encapsulate uncharged solid particle materials, in particular,organic crystalline templates that are largely water insoluble and/orhydrophobic. Thus, the method is applicable to a wide variety ofsubstances and in particular to substances which are of highpharmaceutical interest.

In the first step of the process the uncharged solid particle materialsare treated with an amphiphilic substance, which imparts charge on thesurface of the particle materials. Then in the second step the materialcoated with amphiphilic substance is coated again with a polyelectrolytewhich is oppositely charged to the surface of the coated particlematerials. For the formation of multilayers the material is subsequentlytreated with oppositely charged polyelectrolytes, i.e. alternately withcationic and anionic polyelectrolytes. Polymer layers self-assemble ontothe pre-charged solid templates by means of electrostatic layer-by-layerdeposition, thus forming a multilayered polymeric shell around the solidcores.

Due to the semi-permeable nature of the polymer multilayer shell, it isfurther possible to remove the templated solid core, e.g. by exposure toa mild organic solvent. The process according to the invention thusprovides a novel and facile pathway to the fabrication of polymermultilayered microcapsules as well as superior strategy for theencapsulation of hydrophobic compounds such as drugs.

The limited potential of medical drugs is associated with their lowsolubility in aqueous solutions. Most drugs are solid crystallinesubstances, containing non-polar aromatic groups (amphetamines) and/orheterocyclic groups (1,4-benzodiazepime), or condensed aromatic oralicyclic groups (isoprenoids: steroid, vitamin A, vitamin E) and mostlyone or more polar functional group (e.g. amine, hydroxy, carboxy,phenolic, aldehyde, ketone). Their formulation is a key factor forallowing their use in the human body.

The here-described method provides a strategy for:

-   A) The encapsulation of water-insoluble, uncharged solid particle    material, e.g. drug crystals or/and amorphous (glassy) materials;-   B) The production of hollow polymer capsules.

A) The starting material is the solid substance itself. Due to theuncharged and hydrophobic character of these materials, they cannot bedirectly coated with polyelectrolytes. The described method makes itpossible to introduce a surface charge to the crystal by treatment witha charged amphiphilic species (e.g. ionic surfactants). This leads tothe formation of a stable suspension of the coated substance in water.Typical surface potentials after treatment with an amphiphile (e.g.sodium dodecyl sulfate, SDS) are between −50 and −70 mV, indicating asuspension of surfactant-charged crystals of high stability. The chargedcrystals are then suitable templates for coating with polyelectrolytes.

One advantage of this innovation is the possibility to create a drugrelease system with a constant release rate over a long time period.This is possible because of the solid phase in the interior of thecapsule. After the encapsulated solid material is applied in a liquidsuch as a buffer or body fluid, a two-phase system will be establishedby the crystal itself and a saturated solution of the crystal materialin the interior of the capsule. By contacting the capsules preparedaccording to the invention, which consist of an encapsulated solidmaterial, with a liquid such as water, the liquid penetrates into thecapsules. Thereby the wall of the capsule swells to some degree andwithin the capsule a small portion of the solid material is solubilizeduntil saturation of the penetrated liquid is reached. As release of thematerial occurs, the amount of substance released from the capsule iscontinuously replenished by further solubilization of solid materialwithin the capsule. Therefore the concentration of the substance in theliquid within the capsule remains almost constant. Consequently, aconstant release of the substance over a long period of time can beachieved.

The process of the invention is particularly suitable to prepare releasesystems, which release a small amount of active substance constantlyover an extended period of time. Such a system advantageously comprisesa high diffusion barrier across the wall of the capsule, which resultsin a small amount of released substance with respect to the amount ofsubstance which can be supplemented in the same period of time bysolubilization of solid material within the capsule. Such releasesystems are particularly useful in hormone therapy wherein the constantrelease of small amounts of active substance is required.

The release rate of a substance is a function of the difference in itsconcentration in and outside of the capsule. The described approachprovides a method to keep this concentration gradient constant as longas the solid material is not dissolved completely. This would lead to aconstant release rate of the substance over a long time period. Thismethod provides an advantage over other release systems that use adissolved substance in a capsule or liposome. The concentration of thedissolved substance in those capsules or liposomes is decreased from thefirst moment of the release, and the release rate is not constant.

B) The method shows also the possibility for the production of hollowpolymer capsules. Until now the template removal process has employedharsh conditions (e.g. pH<1.6, pH>11), in order to decompose thepolymeric-template and to remove it out of the capsule. Using theseharsh procedures the capsule itself in many cases is damaged and changedin its properties. This problem can be overcome by using a nonpolymer-template like a crystallized, hydrophobic low molecular weightsubstance (as explained above). This substance can be easily removedafter encapsulation by treatment with a mild organic solvent (e.g.ethanol). The substance is dissolved and readily penetrates through thepolymer multilayers. The shells can then be pelleted by centrifugationand resuspended in an aqueous solution.

According to the invention a solid uncharged material is encapsulated intwo steps. First, the solid uncharged material is treated with anamphiphilic substance. This treatment preferably results in an aqueousdispersion of the solid material. The amphiphilic substance is arrangedon the surface of the solid material, rendering the material susceptibleto the subsequent coating with a charged polyelectrolyte. The unchargedmaterial used in the present invention is preferably a material whichhas no charges and also has no ionizable groups. It is also possible,however, to use an uncharged material having ionizable groups, wherebythis material can be used under conditions, under which the ionizablegroups are not ionized. The encapsulation process according to theinvention is therefore applicable to a wide range of materials, whoseencapsulation in a solid form has previously not been possible orpossible only under very specific conditions. In a second step, thesolid particle material having the amphiphilic substance assembled onits surface is coated with a layer of a charged polyelectrolyte or witha multilayer comprising alternating layers of oppositely chargedpolylectrolytes. The coating of the charged polyelectrolyte onto thesurface is made possible by the amphiphilic substance. By successivetreatment with oppositely charged polyelectrolytes multilayer coatingscan be prepared. Preferably, capsules are prepared having at least two,more preferably at least three, still more preferably at least five andmost preferably at least eight layers of polyelectrolytes havingalternating charge. However, it is also possible to prepare thickershells having e.g. up to 100 or more polyelectrolyte layers, preferablyup to 50 and most preferably up to 20 layers. The assembly of thickershells has the effect of smoothing out the outer surface and at the sametime reducing the porosity of the shells.

By the number of polyelectrolyte layers, by selecting the amphiphilicsubstance and the polyelectrolytes used and by the conditions duringcoating with the amphiphilic substance the porosity of the capsules canbe influenced. In this way, pore sizes specifically designed for therespective application can be obtained. Monomeric detergents such asSDS, for example, lead to small pores, whereas by polymeric detergentssuch as PSS larger pores are obtained in the capsule wall. Theconditions applied when charging the solid material with the amphiphilicsubstance can influence pore size, e.g., if polymeric detergents areused, by determining the shape of the polymeric detergent. Ionicstrength and pH value, for example, can determine whether the polymericdetergent is present in elongated or coiled form. For example, poreshaving a diameter of about 20 nm to >100 nm can be obtained in thecapsule walls, if an amphiphilic polyelectrolyte is used as theamphiphilic substance. However, if an ionic surfactant is used as theamphiphilic substance, smaller pore sizes of less than about 5 to 10 nmcan be obtained. The porosity of the capsule can be decreased by afurther cross-linking step, in which a reagent is used to introduceinter- and intra-crosslinks of polyelectrolytes in the shells.

The thickness of the capsule shell preferably is about 2 to 100 nm, morepreferably 5 to 50 nm. The size of the capsules themselves preferably is<50 μm, in particular, <20 μm and more preferably <15 μm; however, it isalso possible to prepare larger capsules. The minimum size of thecapsules preferably is at least 10 nm, more preferably at least 50 nm.The capsule size basically depends on the size of the solid materialused.

The method of the invention is especially suitable for uncharged solidmaterial which has a low solubility in water or is water-insoluble ornot dispersible in water. The encapsulation of such materials wasdifficult in the prior art and can now be managed easily according tothe invention.

The uncharged solid material used as core for the encapsulation can bean organic material, a biomaterial or/and an inorganic material. Organicmaterials, in particular, solid materials from low-molecular weightorganic compounds can be encapsulated especially favorably. According tothe invention, encapsulation of uncharged solid organic crystallinetemplates that are largely water-insoluble is possible. Suitablematerials which can be encapsulated according to the method of theinvention, for example, are drugs, vitamins, nutrients, hormones, growthfactors, pesticides, antibiotics and preservatives. According to theinvention it is not necessary thereby for the materials to have acharged or ionizable group.

The shape of the capsules largely depends on the shape of the solidmaterial used. Suitably, the solid material is employed as crystallinematerial, e.g. in the form of single crystals, as amorphous orlyophilized materials, spray-dried materials and/or milled materials. Itis particularly preferred to use microcrystals of the unchargedcompounds to be encapsulated. Basically, any uncharged solid materialcan be encapsulated, e.g. a synthetic material, a material isolated fromnatural sources or a chemically modified isolated material.

As amphiphilic substance according to the invention any substance can beused which has ionic hydrophilic and hydrophobic groups. It is importantthat the amphiphilic substance has at least one electrically chargedgroup to provide the solid material with electrical charges. Therefore,the amphiphilic substance used also can be referred to as ionicamphiphilic substance or ionic detergent. Preferably, ionic surfactants,phospholipids and/or amphiphilic polyelectrolytes are used. Amphiphilicpolyelectrolytes, for example, are polyelectrolytes comprising a chargedgroup as hydrophilic group and a hydrophobic group, e.g. aromaticgroups. It is preferred to use a cationic or/and anionic surfactant.Examples of suitable cationic surfactants are quaternary ammonium salts(R₄N⁺X⁻), especially didodecyldimethylammonium bromide (DDDAB),alkyltrimethylammonium bromides, especially dodecyltrimethylammoniumbromide or palmityl trimethylammonium bromide or N-alkylpyridinium saltsor tertiary amines (R₃NH⁺)X⁻), especiallycholesteryl-3β-N-(dimethyl-aminoethyl)-carbamate or mixtures thereof,wherein X⁻ means a counteranion, e.g. a halogenide. Examples of suitableanionic surfactants are alkyl sulfonate (R—SO₃M), especially dodecylsulfate, e.g. sodium dodecyl sulfate (SDS), lauryl sulfate or olefinsulfonate (R—SO₃M), especially sodium-n-dodecyl-benzene sulfonate oralkyl sulfates (R—OSO₃M) or fatty acids (R—COOM), especially dodecanoicacid sodium salt or phosphoric acid or cholic acids or fluoroorganics,especially lithium-3-[2-(perfluoroalkyl)ethylthio]propionate or mixturesthereof. Particularly preferred are surfactants having 1 to 30 carbonsin their alkyl or olefin group.

Further, it is preferred to use a polymeric substance which providescharged groups and hydrophobic sides, in particular, poly(styrenesulfonate) (PSS) as amphiphilic substance.

Polyelectrolytes, generally, are understood as polymers having ionicallydissociable groups, which can be a component or substituent of thepolymer chain. Usually, the number of these ionically dissociable groupsin polyelectrolytes is so large that the polymers in dissociated form(also called polyions) are water-soluble. The term polyelectrolytes isunderstood in this context to cover also ionomers, wherein theconcentration of ionic groups is not sufficient for water-solubility,however, which have sufficient charges for undergoing self-assembly.However, the shell preferably comprises “true” polyelectrolytes, i.e.water-soluble polyelectrolytes.

Depending on the kind of dissociable groups polyelectrolytes areclassified as polyacids and polybases.

When dissociated polyacids form polyanions, with protons being splitoff, which can be inorganic, organic and biopolymers. Examples ofpolyacids are polyphosphoric acid, polyvinylsulfuric acid,polyvinylsulfonic acid, polyvinylphosphonic acid and polyacrylic acid.Examples of the corresponding salts which are also called polysalts, arepolyphosphate, polysulfate, polysulfonate, polyphosphonate andpolyacrylate.

Polybases contain groups which are capable of accepting protons, e.g. byreaction with acids, with a salt being formed. Examples of polybaseshaving dissociable groups within their backbone and/or side groups arepolyallylamine, polyethylimine, polyvinylamine and polyvinylpyridine. Byaccepting protons polybases form polycations.

Suitable polyelectrolytes according to the invention are alsobiopolymers such as alginic acid, gummi arabicum, nucleic acids,pectins, proteins and others as well as chemically modified biopolymerssuch as carboxymethyl cellulose and lignin sulfonates as well assynthetic polymers such as polymethacryl acid, polyvinylsulfonic acid,polyvinylphosphonic acid and polyethylenimine.

Linear or branched polyelectrolytes can be used. Using branchedpolyelectrolytes leads to less compact polyelectrolyte multilayershaving a higher degree of wall porosity. To increase capsule stabilitypolyelectrolyte molecules can be crosslinked within or/and between theindividual layers, e.g. by crosslinking amino groups with aldehydes.Furthermore, amphiphilic polyelectrolytes, e.g. amphiphilic block orrandom copolymers having partial polyelectrolyte character, can be usedto reduce permeability towards polar small molecules. Such amphiphiliccopolymers consist of units having different functionality, e.g. acidicor basic units, on the one hand, and hydrophobic units, on the otherhand, such as styrenes, dienes or siloxanes which can be present in thepolymer as blocks or distributed statistically. By using copolymerswhich due to outside conditions change their structure the permeabilityor other properties of the capsule walls can be controlled in a definedmanner. In this context, for example, copolymers having apoly(N-isopropyl-acrylamide) part, e.g.poly(N-isopropylacrylamide-acrylic acid) are possible which, via theequilibrium of hydrogen bonds, change their water solubility as afunction of temperature, which is accompanied by swelling.

By using polyelectrolytes which are degradable under certain conditions,e.g. photo-, acid- or base-labile, the release of enclosed activesubstance can be further controlled via the dissolution of the capsulewalls. Further, for certain applications, conductive polyelectrolytes orpolyelectrolytes having optically active groups can be used as capsulecomponents. Basically, there are no limitations with regard to thepolyelectrolytes and ionomers, respectively, to be used, as long as themolecules used have sufficiently high charge or/and are capable ofbinding with the layer beneath via other kinds of interaction, e.g.hydrogen bonds and/or hydrophobic interactions.

Suitable polyelectrolytes, thus, are both low-molecular polyelectrolytesand polyions, respectively, e.g. having molecular weights of a fewhundred Daltons, up to macromolecular polyelectrolytes, e.g.polyelectrolytes of biological origin, having a molecular weight ofseveral million Daltons.

Further examples of an organic polymer as bioelectrolyte arebio-degradable polymers such as polyglycolic acid (PGA), polylactic acid(PLA), polyamides, poly-2-hydroxy-butyrate (PHB), polycaprolactone(PCL), poly(lactic-co-glycolic)acid (PLGA), fluorescent-labelledpolymers, conducting polymers, liquid crystal polymers, photocontactingpolymers, photochromic polymers and their copolymers and/or mixturesthereof.

Examples of biopolymers preferred as polyelectrolyte are polyaminoacids, in particular, peptides, S-layer proteins, polycarbohydrates suchas dextrin, pectin, alginate, glycogen, amylose, chitin, chondroitin,hyarulonic acid, polynucleotides, such as DNA, RNA, oligonucleotidesor/and modified biopolymers such carboxymethyl cellulose, carboxymethyldextran or lignin sulfonates. Preferred examples of inorganic polymersas polyelectrolyte are polysilanes, polysilanoles, polyphosphazenes,polysulfazenes, polysulfides and/or polyphosphates.

It is also possible to deposit charged nanoparticles or biomolecules ascapsule material.

The method of the invention preferably is carried out so that excessmaterial of the starting substances used in the individual steps areseparated after each treatment step. For example, an aqueous dispersionof the template particles is formed first by adding an aqueous solutionof the amphiphilic substance. After separating any excess amphiphilicmolecules a first polyelectrolyte species is then added to build up thefirst polyelectrolyte shell. After separating any excess polyelectrolytemolecules the oppositely charged polyelectrolyte species used forbuilding up the next layer is then added. Subsequently, oppositelycharged layers of polyelectrolyte molecules are applied in turn. It ispossible to select identical or different polyelectrolyte species ormixtures of polyelectrolyte species for each layer having the samecharge, i.e. every second layer. Between each incubation step apurification step is carried out.

The encapsulated material prepared preferably forms a stable suspensionin an aquatic phase.

An advantage of the invention lies in that the capsule thickness andpermeability for the controlled release of the encapsulated material canbe controlled in a predetermined manner, e.g. by the number of layers,the nature of the polyelectrolytes used, the nature of the amphiphilicsubstances used, the nature of the nanoparticles or biomolecules, ifused, an optional additional cross-linking step and conditions ofpolyelectrolyte assembly.

After the desired number of polyelectrolyte layers has been appliedaccording to the invention the now encapsulated template particles canbe disintegrated, if desired, leading to the formation of hollowcapsules. The invention, therefore, also encompasses a process for thepreparation of hollow capsules having a polyelectrolyte shell,comprising the steps: (a) treating an uncharged solid particle materialwith an amphiphilic substance, (b) subsequently coating the solidmaterial with a layer of a charged polyelectrolyte or with a multilayercomprising alternating layers of oppositely charged polyelectrolytes and(c) removing the core of uncharged solid particle material by itssolubilization.

Disintegration can be effected by adding reagents which are suitable fordissolving the uncharged solid core material, e.g. an organic solvent,preferably a mild organic solvent, in which the material is soluble oran acid or alkaline solvent in which the material forms a soluble salt.The organic solvent can be used in anhydrous, pure form or asH₂O/solvent mixtures. Representatives of suitable organic solvents aree.g. ethanol, chloroform etc. According to the invention dissolution ofthe template particles can be effected in a gentle manner during a shortincubation period, e.g. 1 min to 1 h at room temperature. The templatesdisintegrate almost completely, as no residues of the particles can bedetected any longer even when inspecting the remaining shells by anelectron microscope.

Preferably, the hollow capsules are redispersed in an aqueous solvent orin an organic solvent. Inside the capsules there is then preferably puresolvent.

Another subject matter of the present invention are polyelectrolytecapsules obtainable by the method of the invention. In one embodiment,these capsules contain a core of uncharged solid material which servedas template. The structure of such capsules thus, viewed from inside tooutside, consists of the following layers: active substance, amphiphilicsubstance and one or more layers of polyelectrolyte. In anotherembodiment, the polyelectrolyte capsules have no detectable residues ofthe uncharged solid core material any longer, i.e. they are withoutcore. Such hollow polyelectrolyte capsule has the following structure:hollow space, amphiphilic substance, one or more layers ofpolyelectrolyte. The advantage of the amphiphilic material contained inthe polyelectrolyte capsules is that porosity can be controlled anddetermined thereby. Furthermore, by using the amphiphilic substance aneven covering of the core material is achieved so that thepolyelectrolyte capsules preferably have an outer shape determined bythe core. It is especially preferred that the polyelectrolyte capsulesaccording to the invention contain an active substance, especially apharmaceutically active substance. The encapsulated active substancethereby can be identical to the encapsulated uncharged solid particlematerial, however, it may also have been introduced into the emptypolyelectrolyte shells later on.

The capsules according to the invention preferably have a diameter inthe range of from 10 nm to 50 μm, preferably from 50 nm to 10 μm. Bysuitable selection of the templates capsule compositions can be obtainedhaving high monodispersity, i.e. compositions, wherein the amount ofcapsules, the deviation of which from the mean diameter is >50%, is lessthan 10% and preferably less than 1%. The capsules according to theinvention also can be dried, in particular, freeze-dried and thenredispersed in suitable solvents again.

It was surprisingly found that the type of amphiphile used to pre-chargethe solid material, in particular, microcrystals, determined theporosity of the resulting capsules. Thus, it is possible to provideunique, highly flexible systems with tailored release properties forencapsulated substances, in particular, for drug delivery applications.For influencing porosity it is also possible to store amphiphilicsubstances, in particular, phospholipids, ionic surfactants oramphiphilic polyelectrolytes between the polyelectrolyte shells.

The capsules prepared by the method of the invention can be used forencapsulating active substance. These active substances can be bothinorganic and organic substances. Examples of such active substances arecatalysts, in particular, enyzmes, pharmaceutically active substances,polymers, colorants such as fluorescent compounds, sensor molecules,i.e. molecules reacting detectably to the change of ambient conditionssuch as temperature or pH, plant protection agents and aromatics. Theactive substances thereby can form the encapsulated uncharged solidmaterials themselves, or be introduced subsequently into the hollowpolyelectrolyte shells obtained by dissolving the core under mildconditions, e.g. by means of an organic solvent.

The capsules also can be used as reaction chambers, especially asmicroreaction chambers, for chemical reactions. Due to the fact that thepermeability of the capsule walls is controllable so as to let pass, forexample, low-molecular substances, however, largely retainmacromolecular molecules, high-molecular products forming in a reaction,e.g. polymers forming upon polymerization, can be retained easily in theinterior during synthesis.

The capsules also can be used in a variety of other applications, e.g.in sensorics, surface analytics, pharmacy, medicine, food technology,biotechnology, cosmetics, information technology and printing industry(e.g. encapsulation of coloring materials).

In the following the invention is explained in detail by the two modelsubstances: pyrene (PYR) and fluoresceine diacetate (FDA), however, itcan also be carried out in general with other uncharged solid materials.

Pyrene (PYR) and fluorescein diacetate (FDA) were employed as theuncharged microcrystalline templates. Both PYR and FDA have a very lowsolubility in water. The first and significant step in the encapsulationinvolved imparting a charge on the crystal surface by self-assembly ofan amphiphilic substance, in particular a ionic surfactant, aphospholipid or polyelectrolyte having an amphiphilic nature such ascharged polymer that is amphiphatic. Preferably, the micrometer-sizedcrystals were dispersed in water, e.g. by sonicating them in thepresence of ionic surfactant.²⁰ The amphiphilic film stabilizes themicrocrystal by both hydrophobic and hydrophilic interactions, coatingand enveloping it and thus protecting it from aggregation. The stableand charged microcrystals coated with the amphiphilic substance, inparticular, a charged surfactant, were then exposed to polyelectrolyte(bearing an opposite charge to the amphiphilic substance adsorbed on thecrystalline template), resulting in their additional coating with apolymer layer. Subsequent consecutive adsorption of oppositely chargedpolyelectrolytes resulted in the formation of polymer multilayers on themicrocrystal colloidal core.²¹

Thus, the assembly of polymer multilayers onto the coated microcrystaltemplates can be achieved by a layer-by-layer adsorption of cationic andanionic polyelectrolytes.

FIG. 2 shows the ζ-potential as a function of the polymer coating layernumber for PYR and FDA microcrystals pre-exposed to surfactant (DDDAB orSDS, FIG. 2 a), DPPC or PSS (FIG. 2 b). PYR crystals exposed to DDDAB(positively charged) showed a ζ-potential of +50 mV, while SDS(negatively charged) dispersed FDA crystals exhibited a value of −50 mV.Furthermore, FDA microcrystals dispersed with DPPC yielded a ζ-potentialof +20 mV and those exposed to PSS a value of −40 mV. These data confirmcharging of the microcrystal surface through adsorption of theamphiphilic substances or PSS, explaining the dispersability of themicrocrystals in aqueous solution. The adsorbed layer coats themicrocrystal, thus protecting it from aggregation.

The mechanism of microcrystal dispersion and stabilization can beexplained by the hydrophobic interactions between the amphiphiles andthe microcrystals. Since both PYR and FDA are hydrophobic, thehydrophobic chains of the surfactants and those on DPPC are expected tobe associated with the microcrystal surface, while the ionic groups onthese amphiphiles project away from the surface.²⁶ It is worthy to notethat neither the PYR nor FDA microcrystals could be readily dispersedwith the polyelectrolytes PAH, poly(diallyldimethylammonium chloride)(PDADMAC), or copolymers of DADMAC and acrylamide with varying DADMACcontents (8–73 mol %). In contrast, the microcrystals could be dispersedby exposure to PSS. The amphiphilic nature of PSS, owing to the aromaticgroup on the polymer backbone (and the charged groups), may beresponsible for the successful adsorption and consequent charging of thecrystal surface. The coated microcrystals are prevented from furthergrowth (i.e. aggregation) by the ionic and/or steric interactions of thethin coating that is tightly associated with each microcrystal particle.These surface modified microcrystals represent stable and chargedcolloids suitable for polyelectrolyte multilayer coating.

As depicted in FIG. 2, an alternating sign in the ζ-potential wasobserved when the pre-charged crystals were exposed to polymer solutionsof opposite charge. The sign of the ζ-potential depended on thepolyelectrolyte that formed the outermost layer, i.e. the polymer thatwas deposited. Regardless of the microcrystal type (PYR or FDA), or theamphiphile used to coat and stabilize the microcrystals, alternatingpositive and negative ζ-potentials were measured for coated crystalsalternately exposed to PAH and PSS, respectively. This shows thatstep-wise growth of the polymers on the microcrystal template occurred,and is characteristic of polymer multilayer formation on chargedcolloidal particles. Values of ca. +50 mV were observed when PAH formedthe outermost layer and −50 mV when PSS was deposited last. The slightlylower positive values observed for the PAH-FITC (FITC: fluoresceinisothiocyanate) layers (ca. +20 mV) is attributed to the high loading ofnegatively charged FITC molecules on the PAH chains. Importantly, theamphiphiles were strongly adsorbed onto the microcrystals allowing theformation of polymer multilayers.

According to the invention, charged surfactants, lipids or amphiphilicpolymers can be used to charge hydrophobic crystalline templates, thusfacilitating their encapsulation with polyelectrolyte multilayers.Surprisingly, the amphiphilic substances are not removed from thesurfaces of the uncharged solid material when a polyelectrolyte isadded, but serve as linkers for the attachment of the polyelectrolyteonto the uncharged solid material.

Additional evidence for the successful encapsulation of the unchargedmicrocrystals was obtained by transmission and fluorescence confocallaser scanning microscopy (CLSM) measurements. CLSM was employed toinvestigate the morphology of the microcrystals and to verify theircoating with polymer multilayers. A fluorescently labelledpolyelectrolyte (FITC-PAH) was adsorbed as the outermost layer on thepre-coated microcrystal colloids in order to allow its visualisation byfluorescence microscopy. The regular coverage of FITC-PAH on the crystalsurface was confirmed by fluorescence microscopy, whilst thetransmission micrographs showed that the coated microcrystal consistedof a solid core. The coated microcrystals could be stored for dayswithout any noticeable change in morphology.

FIG. 3 displays a CLSM image (in transmission mode) of a FDAmicrocrystal that has been dispersed as a result of coating with PSS andadditionally coated with nine polyelectrolytes layers (the last layerwas PAH-FITC). The inset shows the corresponding CLSM fluorescencemicrograph. It is evident from the transmission image the microcrystalpossesses a solid core. The microcrystals had various shapes, rangingfrom near-spherical to rod-like, square and rectangular. Direct evidencefor polymer coating of FDA is provided in the CLSM fluorescence image(inset). This displays fluorescence due to PAH-FITC present in the outerlayer of a coated microcrystal. Similar CLSM images were observed forpre-dispersed FDA and PYR crystals coated with polymer. Studies showedthat the coated microcrystal suspensions were stable for days whenstored in an aqueous medium, reflecting the stability of the adsorbedlayers.

Direct proof that a polymer multilayer cage encapsulated themicrocrystals was obtained by removing the templated core. The releasebehaviour of the pyrene and fluoresceine diacetate molecules, fromdissolution of the core templates, through the polymer capsule wall canbe investigated by using fluorescence spectroscopy. Followingcentrifugation of the coated microcrystal suspensions that were exposedto ethanol, the supernatant was assessed for either pyrene orfluoresceine at regular time intervals.

Control experiments for DDDAB-dispersed PYR microcrystals and thosedispersed with PSS revealed rapid release characteristics: Upon additionof ethanol, the pyrene core was removed within about 30 min for both thesurfactant- and PSS-coated crystals. The pores in the polymermicrocapsules produced in this work are large enough to allow removal ofthe low molecular weight core molecules (see below). This finding isconsistent with earlier reports on the permeability characteristics ofpolyelectrolyte multilayers: Polymer multilayers are permeable to lowmolecular weight substances^(14,15) but essentially impermeable topolymers larger than 4000 Da.²⁷ Further experiments showed that the rateof removal was found to be dependent on the first layer adsorbed, thenumber of polyelectrolyte layers, and the ratio of ethanol to water inthe dissolving medium. It is worth noting that up to fivepolyelectrolyte layer pairs were assembled onto the microcrystaltemplates in the current work. Slower release rates were observed withincreasing polyelectrolyte layer number. The assembly of thicker shells(e.g. more polyelectrolyte layers) may have the effect of smoothing outthe outer surface and at the same time reducing the porosity. Thelayer-by-layer assembly of polycations and polyanions displays aremarkable self-regularity: For films grown on poorly charged and/orrough planar substrates, irregular growth has often been observed forthe first few layers with regular growth achieved after the depositionof a number of layer pairs.³⁰⁻³²

The CLSM micrographs of polymer-coated FDA microcrystals after beingexposed to ethanol solution and dispersed in water are displayed in FIG.4. The transmission image (a) shows a number of the hollow colloidalentities produced. There is no evidence of a solid core, indicatingdissolution and removal of the microcrystal. Ethanol solubilizes thecore material and the individual molecules are then able to diffusethrough the semi-permeable polymer capsule walls. The structures seen inthe transmission image are due to the contrast of the remaining polymerlayers from the original coating of the microcrystals, indicating thesuccessful formation of hollow polymer capsules. The above results areconsistent with the visual observation that the polymer-coatedmicrocrystal suspensions lost their turbidity upon the addition ofethanol. Further evidence is provided by the corresponding CLSMfluorescence image (b), which shows the fluorescence from the PAH-FITClayers. The different morphologies observed are due to the diversity ofthe microcrystal shapes. Some indentations on the polymer capsule wallsmay also be due to the centrifugation process used in their preparation.There was no evidence of rupturing of the capsule walls as a result ofthe facile removal of the microcrystal core by treatment with ethanol.The CLSM results demonstrate that polymer multilayers can be depositedonto pre-charged microcrystal templates and that the core can be removedby treatment with an appropriate solvent, leaving behind hollow polymercapsules.

The polyelectrolyte capsules produced were further characterized usingTEM and AFM. TEM images of air-dried hollow polymer capsules obtainedfrom SDS-dispersed PYR microcrystals coated with eleven polymer layers,and FDA crystals dispersed with PSS and additionally coated with ninepolyelectrolyte layers, are illustrated in FIGS. 5 (a and b,respectively). The insets are higher magnifications. The folds andcreases seen in the polymer capsules are a result of evaporation of theaqueous content by air-drying.¹⁰ The striking difference between FIG. 5a and FIG. 5 b is the wall porosity. Capsules produced when themicrocrystals were dispered with surfactant (either positively ornegatively charged) exhibit a much smoother texture and lower porositythan those produced from PSS-dispersed microcrystals. Pores of diameterfrom 20 nm to larger than 100 nm were observed for hollow capsulesderived from polymer-coated PSS-dispersed microcrystals. In contrast, itwas difficult to discern pores in the very smooth textured polymercapsules when surfactant was used to disperse the microcrystals,suggesting an average pore size of less than about 5–10 nm. Thesefindings were confirmed by AFM measurements. The differences seen may beascribed to the initial conformation of the first adsorbed layer (interms of homogeneity) used to disperse the crystals. Nevertheless, theabove illustrates the importance of the first adsorbed layer indetermining the porosity of the resulting thin-walled hollow polymercapsules. Control over the pore size in such hollow microcapsules isexpected to have important implications in technology as it allowsregulation of the release rate of encapsulated materials.

Examination of the apparent heights of air-dried polymer capsules byusing tapping mode AFM yielded values of approximately 25–30 nm forcapsules comprised of 10 polyelectrolyte layers. This dimension isequivalent to twice the polymer capsule wall thickness; hence, theaverage thickness per polyelectrolyte layer is between 1 to 1.5 nm, avalue that is close to those obtained for polymer multilayers on othercolloidal templates.¹⁰

An attractive feature of the process employed for the formation ofhollow polymer capsules is the facile removal of the templatedmicrocrystal core. Previous methods have involved extremely acidic(pH=1)^(10,15) or basic (>12)^(10,17) solutions. Clearly the use of suchconditions is limited, particularly when biological compounds arepresent during the core removal process. In addition, the undesirablechanges in the composition and properties of the hollow polymer capsulesthat occur with such harsh conditions²⁹ can be avoided.

In summary, the colloid-template approach based on uncharged organicmicrocrystals complements other strategies we have been developing forencapsulating various materials. This method is of particular relevanceand importance because of its potential to encapsulate a wide range ofuncharged crystalline drugs. In addition, its versatility and thecontrol that it permits over the polymer multilayer wall thickness andcomposition allows for the creation of a drug release system with atailored release rate. The systems prepared provide excellent model drugrelease systems to study various parameters on the release rate ofencapsuled low molecular weight compounds. An interesting strategy is tocontrol the release rate by varying the thickness and composition of thepolymer capsule walls.

The invention is further illustrated by the following examples andfigures.

FIG. 1 is a schematic representation of a preferred embodiment of theprocess used to encapsulate organic microcrystals and to create hollowpolymer cages. The uncharged microcrystals are coated by theself-assembly of charged surfactant molecules (step 1), rendering themwater dispersible and hence amenable to subsequent coating withpolyelectrolyte multilayers (step 2). Each polyelectrolyte layerdeposited has an opposite charge to that already adsorbed. Hollowpolymer multilayer cages are formed by direct exposure of theencapsulated microcrystals to ethanol, causing their solubilisation andremoval (step 3). Some surfactant may be electrostatically bound to thehollow polymer cages.

Step 1 of FIG. 1 can, of course, be varied, e.g. by use of apolyelectrolyte (e.g. PSS) or phospholipids to coat and pre-charge themicrocrystals.

FIG. 2 shows the ζ-potential of amphiphile-stabilised PYR and FDAmicrocrystals as a function of polyelectrolyte layer number: (a)PYR-DDDAB (filled squared), FDA-SDS (open circles); (b) FDA-DPPC (filledcircles); FDA-PSS (open squares). Layer number=1 corresponds to theamphiphile-coated microcrystals. Surface charge reversal is seen withadsorption of each polyelectrolyte layer. From layer number 2 onwards,positive values are for PAH adsorption and negative values for PSSdeposition. Layer 9 and 11 for the PYR-DDDAB system and layer 10 for theFDA-PSS system correspond to PAH-FITC adsorption.

FIG. 3 shows the transmission and fluorescence (inset) CLSM micrographsof an FDA crystal dispersed by adsorption of PSS and further coated withnine polyelectrolyte layers, with the outermost layer being PAH-FITC,[(PAH/PSS)₄/PAH-FITC].

FIG. 4 shows the CLSM transmission (a) and fluorescence (b) micographsof hollow polymer capsules derived from polymer-coated FDAmicrocrystals. The polymer cages were obtained from polymer-coated FDAmicrocrystals. The FDA microcrystals were dispersed by adsorption of PSSand additionally coated with nine polyelectrolyte layers with theoutermost layer being PAH-FITC, [(PAH/PSS)₄/PAH-FITC]. The insets showan individual hollow polymer cage, obtained after dissolution of thecore from FDA dispersed with SDS and coated with eleven polyelectrolytelayers [(PAH/PSS)₄/PAH-FITC]. The scale bars in the insets correspond to2 μm.

FIG. 5 shows the TEM images of air-dried hollow polymer capsules,obtained after removal of the templated microcrystal core with ethanol.(a) The PYR core was dispersed by SDS and coated with elevenpolyelectrolyte layers. (b) FDA was dispersed by PSS and coated withnine polyelectrolyte layers. The polymer capsules flattened as a resultof drying and folds and creases are seen. A significant difference inporosity was observed for the polymer capsules, depending on whethersurfactant (a, less porous) or PSS (b, more porous) was used to dispersethe microcrystals. Similar differences were observed for both the PYRand FDA systems. The scale bars in the insets correspond to 200 nm.

EXAMPLES

1. Materials

Pyrene (PYR) was purchased from Aldrich and fluorescein diacetate (FDA)from Sigma. The polycation, poly(allylamine hydrochloride) (PAH), M_(w)15,000, and the polyanion, poly(sodium 4-styrenesulfonate) (PSS), M_(w)70,000, were obtained from Aldrich. The positively charged surfactantsdidodecyldimethylammonium bromide (DDDAB), hexadecyltrimethylammoniumbromide (HDTAB), dodecyltrimethylammonium bromide (DTMAB),myristyltrimethylammonium bromide (MTMAB), and the negatively chargedsurfactant sodium dodecylsulfate (SDS) were all from Aldrich.Dipalmitoyl-DL-α-phosphatidylcholine (DPPC) was purchased from Sigma.All reagents were used as received, except for the PSS, which wasdialyzed against Milli-Q water (M_(w) cut-off 14,000) and lyophilizedbefore use.

2. Preparation of Fluorescein Isothiocyanate Labeled PAH (PAH-FITC):

An aqueous solution of 500 mg PAH in 6 ml water is adjusted to a pH of8.1 with 1 M NaOH. An aqueous solution of 4 mg FITC in 500 μl DMSO isadded to the PAH solution (conjugation ratio FITC/PAH-monomer is 1/500).The mixture is incubated overnight at room temperature and thenfiltrated with a 3 μm filter. The unconjugated FITC is removed from theconjugate by gel filtration over a PD-10 column (Pharmacia). The finalfractions are dialysed against deionised water overnight by using a0.5–2 ml Slide-A-Lizer frame (Pierce) with a cut-off of molecular weightof 3500 dalton. Yield: 25 ml PAH-FITC solution with a concentration of 9mg/ml.

3. Assembly of Polyelectrolyte Multilayers onto Organic Microcrystals

The layer-by-layer assembly of polyelectrolytes onto FDA or PYRmicrocrystals was carried out as follows: 50 mg of finely milled coreparticles (FDA or PYR) were first thoroughly mixed with 12 mL of 0.2–0.4wt % of the dispersing agent (ionic surfactant, lipid or chargedpolymer). Crystalline fluorescein diacetate or pyrene can be milled tofine particles using e.g. a mortar and pestle. However, also advancedball milling procedures can be used.

The crystals were suspended by their immediate sonication for 5 min. Thesuspension was allowed to stand for 30 min, thus allowing the largercrystals to sediment, or gently centrifuged. The turbid whitesupernatant was then extracted, centrifuged and washed several times,and finally resuspended in water.

The resulting microcrystal particles were then layer-by-layer coatedwith PSS and PAH.¹⁸ When positively charged surfactants or DPPC wereused as the first layer, 1 mL of PSS solution (5 mg mL⁻¹, containing 0.5M NaCl) was added first. PAH solution (1 mL of 5 mg mL⁻¹, containing 0.5M NaCl) was added first when PSS or SDS were adsorbed onto themicrocrystals. After an adsorption time of 15 min for PAH or PSSadsorption, the suspension was centrifuged at 3000 g for 5 min. Thesupernatant was then removed and three cycles of water washing andredispersing were applied to remove the excess unadsorbedpolyelectrolyte in solution. Subsequent polyelectrolyte layers, bearingan opposite charge to that already adsorbed on the particle, weredeposited in identical fashion to produce multilayer-coatedmicrocrystals. In some cases, the fluorescently labeled polyelectrolyte,PAH-FITC, was applied (as a 2 mg mL⁻¹ solution containing 0.5 M NaCl) toform a fluorescent layer on the microcrystal surface.

The order of polyelectrolyte coating and the resulting zeta-potentialsare given in Tables 1 and 2 and are also graphically shown in FIG. 2.

TABLE 1 Zeta-potential of fluorescein diacetate crystals as a functionof the layer number and polyelectrolyte Template: Fluorescein diacetateLayer No. Coating Zeta-potential 1 SDS detergent  −850 mV 2 PAH +42.9 mV3 PSS −37.7 mV 4 PAH +51.5 mV 5 PSS −42.2 mV 6 PAH-FTTC +48.2 mV

TABLE 2 Zeta-potential of pyrene crystals in as a function of the layernumber and polyelectrolyte Template: Pyrene Layer No. CoatingZeta-potential 1 SDS detergent −68.5 mV 2 PAH   +59 mV 3 PSS −46.5 mV 4PAH +54.3 mV 5 PSS −37.2 mV 6 PAH-FTTC +47.3 mV

The treatment of the crystals with the SDS solution leads to a highnegative surface charge (see layer No. 1 in Tables 1 and 2). Theresulting suspensions are highly stable and ideally suitable astemplates for the coating with polyelectrolytes. The alternating chargeof the zeta-potential indicates the successful coating. The coating wasalso confirmed by the application of FITC conjugated PAH, and itsvisualisation by fluorescent microscopy.

The morphology of the crystals is not changed during the coatingprocedures. The yield of coated substance is only decreased by a smallloss in the centrifugation/washing step, and can be optimised to highrates, around 98%.

In addition to the previously described experiment, encapsulations underdifferent conditions (surfactant concentration 0.2–0.4 wt %, sonication5–30 min., lower polyelectrolyte concentrations) were also carried outsuccessfully.

4. Release Experiments

12 mL of solvent (ethanol or ethanol/water mixtures) were dispensed into15 mL tubes. 0.1 mL of the coated microcrystal suspension of Example 3was then quickly added to each tube and after defined times (2, 5, 10min etc.) the suspension was centrifuged at 3000 g for 5 min. A portionof the supernatant was removed and tested for the presence of PYR of FDAby fluorescence. For PYR, the fluorescence emission intensity of thesupernatant was measured directly by using an excitation wavelength(λ_(ex)) of 350 nm and monitoring the emission (λ_(em)) at 373 nm. FDAwas first hydrolyzed into fluorescein either by treatment with esteraseor dilute base prior to fluorescence measurement (λ_(ex)=492 nm,λ_(em)=513 nm). As control experiments, the release characteristics ofuncoated particles was also studied as outlined above.

5. Production of Hollow Polymer Capsules by Removing of the HydrophobicTemplate with Organic Solvents

The microcrystal core was removed by exposing 0.2 mL of the coatedparticle suspension of Example 3 to 1 mL of ethanol (or chloroform) andallowing 30 min for core dissolution. The resulting hollow polymercapsules were then centrifuged at 10000 g for 10 min, exposed to ethanolagain, washed a further two times with water, and finally resuspended inwater.

The resuspended shells were analysed by confocal fluorescence microscopydemonstrating that hollow shells are obtained by the method. The shapeof the shells is irregular due to initial shape of the template. Thiscan be optimized or tailored by suitable choice of the template.

6. Instruments and Test Methods

Microelectrophoresis

The microelectrophoretic mobility of coated organic microcrystals wasmeasured with a malvern Zetasizer 4 by taking the average of 5measurements at the stationary level. The mobilities (μ) were convertedto the electrophoretic potentials (ζ) using the Smoluchowski relationζ=μη/ε, where η and ε are the viscosity and permittivity of thesolution, respectively.²⁵ All measurements were performed onmicrocrystals re-dispersed in air-equilibrated pure water (pH ˜5.6).

Confocal Laser Scanning Microscopy (CLSM)

CLSM images were taken on a confocal laser scanning Aristoplanmicroscope from Leica with a 40×oil immersion objective.

Transmission Electron Microscopy (TEM)

TEM measurements were performed on a Philips CM12 microscope operatingat 120 kV. TEM samples were prepared by deposition of a diluted particlesuspension onto a carbon-coated copper grid. The mixture was allowed toair dry for one minute, after which the time exces solution was removedby blotting with filtered paper.

Atomic Force Microscopy (AFM)

AFM images were obtained using a Nanoscope IIIa AFM (DigitalInstruments, CA) in tapping mode. Samples were prepared by applying adrop of a diluted solution onto a freshly cleaved mica surface, allowing1 min for air drying, and then blotting off the extra solution.

Fluorescence Spectroscopy

Steady state fluorescence spectra were recorded using a Spex Fluorolog1680 spectrometer. Both excitation and emission bandwidths were set at1.0 nm. All measurements were performed on air-equilibrated solutions at25° C.

REFERENCES AND NOTES

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Chem. B 1999, 103, 6434.

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1. A process for the encapsulation of an uncharged crystalline solidparticle material comprising: (a) treating the crystalline solidparticle material with an amphiphilic substance and (b) subsequentlycoating the material with a layer of a charged polyelectrolyte or with amultilayer comprising alternating layers of oppositely chargedpolyelectrolytes.
 2. The process according to claim 1, wherein the solidmaterial has a low solubility in water, is water insoluble or notwater-dispersible.
 3. The process of claim 1, wherein the solid materialis an organic material, a bio-material or an inorganic material.
 4. Theprocess according to claim 1, wherein the solid material is selectedfrom the group consisting of drugs, vitamins, nutrients, hormones,growth factors, pesticides, antibiotics and preservatives or mixturesthereof.
 5. The process according to claim 1, where the solid materialis selected from the group consisting of single crystals.
 6. The processaccording to claim 1, wherein the solid material is a synthetic materialor a material isolated from natural sources or a chemical modifiedisolated material.
 7. The process according to claim 1, wherein theamphiphilic substance is selected from ionic surfactants, phospholipidsand amphiphilic polyelectrolytes.
 8. The process according to claim 1,wherein a cationic or an anionic surfactant or a combination of anionicand cationic surfactants is used.
 9. The process according to claim 8,wherein the cationic surfactant is selected from the group consisting ofquarternary ammonium salts ((R₄N⁺)X⁻), alkyltrimethylamoniumbromides,N-alkyl pyridinium salts, tertiary amines, ((R₃NH⁺)X⁻), secondary amines((R₂NH₂ ⁺)X⁻), primary amines ((RNH₃ ⁺)X⁻) and mixtures thereof.
 10. Theprocess according to claim 8, where the anionic surfactant is selectedfrom the group consisting of alkylsulfonates (R—SO₃M), olefinsulfonates(R—SO₃M), alkylsulfates R—OSO₃M, fatty acids (R—COOM), phosphoric acids,cholic acids, fluoro organics, and mixtures thereof.
 11. The processaccording to claim 8, wherein the amphiphilic substance is selected fromthe group consisting of a polymeric substance which provides chargedgroups and hydrophobic sides, and block-copolymers .
 12. The processaccording to claim 1, wherein the polyelectrolyte is selected from thegroup consisting of organic polymers, bio-polymers, inorganic polymersand mixtures thereof.
 13. The process according to claim 1, wherein thepolyelectrolyte is a linear or a non-linear polymer or mixtures thereof.14. The process according to claim 1, wherein the polyelectrolyte is ablock-copolymer.
 15. The process according to claim 1, wherein thepolyelectrolyte is cross-linked after templating.
 16. The processaccording to claim 15, wherein the cross-linking is provided between thepolymers in one layer or/and between the layers.
 17. The processaccording to claim 12, wherein the polyelectrolyte is an organic polymerselected from the group consisting of bio-degradable polymers,fluorescent labelled polymers, conducting polymers, liquid crystalpolymers, photo conducting polymers, photochromic polymers and theircopolymers and mixtures thereof.
 18. The process according to claim 12,wherein the polyelectrolyte is a bio-polymer selected from the groupconsisting of poly amino acids, poly carbohydrates, poly nucleotides,oligonucleotides and modified bio-polymers.
 19. The process according toclaim 12, wherein the polyelectrolyte is an inorganic polymer selectedfrom group consisting of polysilanoles, polysilanoles, polyphosphazenes,polysulfazenes, polysulfides, polyphosphates and mixtures thereof. 20.The process according to claim 1, wherein charged nanoparticles and/orbiomolecules are deposited as capsule materials.
 21. The processaccording to claim 1, wherein excessive material of amphiphilicsubstances, polyelectrolytes and/or nanoparticles and biomolecules, thatare not contributed to forming the coating, are separated after eachcoating step.
 22. The process according to claim 1, wherein theencapsulated material is forming a stable suspension in an aquaticphase.
 23. The process according to claim 1, wherein the capsulethickness and permeability for the controlled release of theencapsulated material is controlled by at least one of the followingfeatures: the nature of the surfactant, the number of layers, the natureof the polyelectrolyte, the nature of the nanoparticles or biomolecules,an additional cross-linking step, the conditions of polyelectrolyteassembly and the nature of amphiphilic coating.
 24. A process for thepreparation of capsules having a polyelectrolyte shell, comprising thesteps: (a) treating an unchared crystalline solid particle material withan amphiphilic substance, (b) subsequently coating the crystalline solidmaterial with a layer of a charged polyelectrolyte or with a multilayercomprising alternating layers of oppositely charged polyelectrolytes and(c) removing the core of uncharged crystalline solid particle material.25. The process according to claim 23, wherein hollow capsules areproduced from the encapsulated material by removal of the core materialby exposure to an organic solvent in which the material is soluble or anacid or alkaline solvent in which the material is forming a soluble saltor mixtures thereof.
 26. The process according to claim 24, wherein thehollow capsules are redispersed in an aqueous solvent or an organicsolvent or mixtures thereof.
 27. The process according to claim 24,wherein further a drug is incorporated into the capsules.
 28. Theprocess according to claim 1, wherein the size of pores within thecapsule wall is controlled by the kind of amphiphilic substance usedand/or the coating conditions of the amphiphilic substance. 29.Polyelectrolyte capsule, obtainable by a process according to claim 1.30. Capsule according to claim 29, comprising a core consisting ofuncharged solid material.
 31. Capsule according to claim 29, comprisingno detectable residue of the solid core material.
 32. Capsule accordingto claim 29, having a final shape which is determined by the unchargedsolid core material.
 33. Capsule according to claim 29, comprising adrug.
 34. A process for preparing a drug-containing capsule, comprisingencapsulating a drug in a polyelectrolyte capsule as claimed in claim29.
 35. A process according to claim 29, wherein said polyelectrolytecapsule is a reaction chamber.
 36. A process according to claim 29,wherein said polyelectrolyte capsule is applied in insensoric,surface-analytic or information technology applications.
 37. A processaccording to claim 29, wherein said polyelectrolyte capsule is appliedin pharmacy, medicine, food technology, biotechnology, cosmetics or inprinting applications.
 38. A process according to claim 29, wherein saidpolyelectrolyte capsule is a slow, targeted, or controlled releasesystems.
 39. Composition containing capsules according to claim 29 indried form.
 40. Composition comprising capsules according to claim 29having a monodisperse size distribution.
 41. The process according toclaim 9, wherein said quaternary ammonium salt isdidodecyldimethylammonium bromide (DDDAB).
 42. The process according toclaim 9, wherein said alkyltrimethyl ammoniumbromide isdodecyltrimethylammonium bromide or palmithyltrimethylammonium bromide.43. The process according to claim 9, wherein said tertiary amine ischolesteryl-3-β-N-(dimethyl-aminoethyl) carbamate.
 44. The processaccording to claim 10, wherein said alkylsulfonate is dodecylsulfate orlaurylsulfate.
 45. The process according to claim 10, wherein saidolefinsulfonate (R—SO₃M) is sodium n-dodecylbenzensulfonate.
 46. Theprocess according to claim 10, wherein said fatty acid (R—COOH) isdodecanoic acid sodium salt.
 47. The process according to claim 10,wherein said fluoro organic is lithium 3-propionate.
 48. The processaccording to claim 11, wherein said polymeric substance ispoly(styrenesulfonate) (PSS).
 49. The process according to claim 11,wherein said block-copolymer is poly(ethylethylene-block-styrene sulfoicacid (PEE-PSS).
 50. The process according to claim 17, wherein saidbiodegradable polymer is polyglycolic acid (PGA), polyactic acid (PLA),polyamide, poly-2-hydroxy butyrate (PHB), polycaprolactone (PCL), orpoly (lactic-co-glycolic) acid (PLGA).
 51. The process according toclaim 18, wherein said polyamino acid is a peptide or a S-layer protein.52. The process according to claim 18, wherein said polycarbohydrate isdextrin, pectin, alginate, glycogen, amylose, chitin, chondroitin, orhyarulonic acid.
 53. The process according to claim 18, wherein saidpolynucleotide is DNA or RNA.
 54. The process according to claim 18,wherein said modified bio-polymer is carboxymethyl cellulose,carboxymethyl dextran, or lignin sulfonate.